1. Field of the Invention
The present invention relates to the fields of protein molecular biology and pathogenic microbiology. More specifically, the present invention relates to BBK32 peptides effective to induce superfibronectin aggregation and uses to treat endothelial cell proliferative-associated diseases or disorders.
2. Description of the Related Art
Borrelia burgdorferi sensu lato is the causative agent of borrelioses such as Lyme disease, the most common arthropod-borne infectious disease in North America and Europe (1). B. burgdorferi has a complicated life cycle and oscillates between ticks and vertebrate hosts. The molecular pathogenesis of Lyme disease is unclear especially since Borrelia does not produce any known virulence determinants or toxins that share homology with other pathogens. It has been suggested that the various lipoproteins on the outer membrane sheath allow the spirochete to interact with host tissues and likely play key roles in the infectious process (2).
Transcriptional analyses of B. burgdorferi under different conditions suggest that a set of 11 lipoproteins are selectively upregulated when the spirochete is exposed to a mammalian host (3). BBK32, a fibronectin-binding 47 kDa MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecule), is one of the upregulated lipoproteins. It was originally identified as a fibronectin-binding MSCRAMM by probing lysates of B. burgdorferi with fibronectin in Far Western blots (2). BBK32 was localized to the surface of the spirochete and the attachment of B. burgdorferi to fibronectin substrates was blocked by addition of soluble recombinant BBK32 protein suggesting that BBK32 is the primary fibronectin-binding adhesin on B. burgdorferi sensu stricto (2).
Orthologous genes are found in the closely related species, B. garinii and B. afzelii (4). Expression of BBK32 at the site of experimental infection in mice increases from days 3 to 7 and then declines, but bbk32 gene expression can be detected in the skin, heart, spleen, joints and bladder at least 30 days post challenge, indicating that the lipoprotein is expressed by the spirochete as it disseminates to different tissues in the host (3). B. burgdorferi mutant strains containing bbk32 deletions have an attenuated infectivity when assessed at twenty-one days post infection (5) demonstrating that BBK32 is a virulence factor in borrelioses.
The N-terminus of BBK32 contains a signal peptide followed by a “lipobox” and an extended disordered segment, residues 21-205, that contains the fibronectin-binding sites (6). The C-terminal part of the protein (residues 206-354) appears as a globular domain that may bind fibrinogen (6). Upon binding to the N-terminal segment of fibronectin, the ligand binding segment of BBK32 undergoes conformational changes to form beta-strands that complement existing beta-sheets in fibronectin modules. This binding mechanism called the Tandem Beta-Zipper mechanism (6-7) has previously been demonstrated for the interaction of fibronectin with MSCRAMMs from Staphylococcus aureus and Streptococcus pyogenes (8).
Fibronectin is a large dimeric glycoprotein found in a soluble form in plasma and other body fluids and in the insoluble fibrillar form in the extracellular matrix. This dynamic protein plays key roles in basic physiological processes such as cell proliferation, migration, and survival by interacting with a variety of extracellular macromolecules and cellular receptors, primarily of the integrin family (9). Fibronectin is composed of three types of repeating modules: 12 type I modules (F1), 2 type II modules (F2), and 15-17 (dependent on alternative splicing) type III modules (F3), and an alternatively spliced variable sequence that is not homologous to other parts of fibronectin. F1 and F2 modules are stabilized by disulfide bonds whereas F3 modules lack disulfide bonds and can reversibly partially unfold (10-11). The two monomers are connected at the C-termini by two disulfide bonds.
Soluble fibronectin adopts a somewhat compact form that is stabilized by intramolecular ionic interactions between specific modules. The interactions occur primarily between the 1F1-5F1, the 2F3-3F3, and 12F3-14F3 segments (12). In the extracellular matrix fibronectin takes on a more extended form and the protein is likely engaged in multiple intra- and intermolecular interactions (13), but the structural organizations of fibronectin matrices have not been elucidated in detail. It is likely that the incorporation of soluble fibronectin into a matrix involves a complex but orderly breaking of intramolecular bonds in the compact soluble fibronectin and the formation of new intra- and intermolecular interactions whereby an extended form of fibronectin is deposited in a matrix. This process is facilitated by integrins and other cellular components (14-15). The fibronectin-binding MSCRAMM Sfb1 from S. pyogenes apparently can interfere in this process and inhibits fibronectin matrix formation in fibroblast cultures (16).
Fibronectin may occur in different aggregated forms. “Superfibronectin” (sFn) is a form of fibronectin aggregates that resembles matrix fibronectin but has distinctly different biological activities (17). Superfibronectin is formed by mixing plasma fibronectin with Anastellin, a recombinant form of the C-terminal two-thirds of 1F3. F3 modules are beta-sandwiches composed of a quadruple-stranded beta-sheet and a triple-stranded beta-sheet. In Anastellin, strands A and B are removed, exposing the E and C strands. This exposure is perhaps responsible for Anastellin's self-association and interaction with other fibronectin modules (18) (1F1.5F1, gelatin binding domain, F3 modules). These interactions lead to the formation of sFn. Ohashi and Erickson recently suggested a model of sFn formation whereby Anastellin binds to partially unfolded F3 modules and prevents refolding, thus exposing hydrophobic surfaces and beta-sheet edges. The exposed elements bind to similar exposed elements on other fibronectin molecules leading to a specific ordered aggregation (19).
A large number of pathogenic microorganisms have been shown to express fibronectin-binding MSCRAMMs although only a few of these interactions have been characterized in detail. The S. aureus fibronectin-binding MSCRAMM FnbpA is composed of 11 repeats that each may bind to the N-terminal 1F1-5F1 segment of fibronectin (8). Sfb1 from S. pyogenes also contains repeats that bind to the N-terminal F1 modules. However, this MSCRAMM also interacts with the gelatin binding domain of fibronectin composed of 6F11F22F27F28F19F1 (20). In mapping the binding sites in fibronectin for BBK32, it was determined that the MSCRAMM not only binds to the N-terminal domain and the gelatin binding domain but also the 1F3, 1-2F3, and 3F3 modules.
Thus, a recognized need is present in the art for alternate methods of inducing superfibronectin aggregation. Specifically, the prior art is deficient in methods of inducing superfibronectin aggregation by contacting fibronectin with BBK32 peptides and in methods of using BBK32-induced superfibronectin aggregates for treating endothelial cell proliferative-associated pathophysiological conditions. The present invention fulfils this longstanding need in the art.